Br. J. clin. Pharmac. (1978), 5, 167-173

THE EFFECTS OF CHRONIC DRUG ADMINISTRATION ON HEPATIC ENZYME INDUCTION AND FOLATE METABOLISM D. LABADARIOS*,J.W.T. DICKERSON & D.V. PARKE Department of Biochemistry, University of Surrey, Guildford, Surrey

E.G. LUCASt & G.H. OBUWAt Brookwood Hospital, Woking, Surrey

I Patients on prolonged treatment with anticonvulsant and phenothiazine drugs exhibited lower than normal concentrations of folate in serum and erythrocytes, and showed increased urinary FIGLU excretion after histidine loading; urinary excretion of D-glucaric acid was also increased suggesting induction of the hepatic microsomal enzymes. 2 Folate deficiency by enzyme-inducing drugs was seen to be determined more by the duration of therapy than by the nature of the drugs. Excretion of FIGLU was increased by 70% by 2-5 years of treatment with anticonvulsant, phenothiazine or tricyclic drugs, and by 200% after 6 or more years. 3 Hepatic microsomal enzyme induction, as measured by D-glucaric acid excretion, was greatest after 2-5 years treatment. 4 It is suggested that the increased requirements for folate, resulting from microsomal enzyme induction, lead to folate deficiency and this subsequently limits enzyme induction, leading to adverse drug side-affects. S The dietary folate of hospitalized patients would seem to be generally inadequate for patients on long term treatment with enzyme-inducing drugs.

Introduction

Prolonged administration of certain anticonvulsant drugs to patients with epilepsy has been shown to be associated with low concentrations of folate in the serum, red blood cells and cerebrospinal fluid (Reynolds, Milner, Matthews & Chanarin, 1966; Reynolds, 1968; Mantz, Tempe & Jaeger, 1970). Explanations of this deficiency have included suggestions that the long-term administration of these drugs (a) interferes with the biosynthesis of folate co-enzymes (Hawkins & Meynell, 1954), (b) impairs the absorption of folate (Meynell, 1966; Dahlke & MertensRosler, 1967), (c) inhibits intestinal conjugases leading to polyglutamate malabsorption (Hoffbrand & Necheles, 1968; Rosenberg, Streiff & Godwin, 1968) and (d) increases the requirement for folate as a consequence of hepatic enzyme induction (Maxwell, Hunter, Stewart & Williams, 1972). Folate is important in the biosynthesis of cytochromes and may also be a co-factor for certain hepatic microsomal hydroxylation reactions (Tietz,

Lindberg & Kennedy, 1964; Hagerman, 1964). Induction of hepatic microsomal enzymes by prolonged administration of anticonvulsant drugs may therefore lead to increased folate requirements, either for the increased de novo synthesis of cytochrome P-450 and the other microsomal enzymes, or for increased cofactor requirements. Further effects consequential upon the induction of liver enzymes by prolonged administration of anticonvulsant drugs include the accelerated turnover of cortisol (Richens, 1976) and an increased rate of deactivation of vitamin D, with accompanying osteomalacia (Hunter, 1976). The present studies were undertaken to investigate the requirements of folate after prolonged administration of anticonvulsants, tricyclic antidepressants and phenothiazines to patients, and the effects of such treatment on hepatic enzyme induction.

Methods Present addresses: *National Research Institute for Nutritional Diseases, P.O. Box 70, Tygerberg 7505, Republic of South Africa

tKing's College Hospital, London, S.E.5 tChainama Hills Hospital, P.O. Box 43, Lusaka, Zambia

The patients and controls were long stay patients at Brookwood Hospital, Surrey. Details of their age, sex, diagnosis, and duration of hospitalization and treatment are given in Table 1. The specific drug regimes

168

D. LABADARIOS, J.W.T. DICKERSON, D.V. PARKE, E.G. LUCAS & G.H. OBUWA

were: (a) anticonvulsants: phenytoin and phenobarbitone, alone or in association with primidone or diazepam; (b) phenothiazines: thioridazine or chlorpromazine; and (c) tricyclics: imipramine or amitriptyline, and the diazepines, nitrazepam or diazepam. No patient was receiving vitamin supplements. Fasting blood samples were taken by venepuncture, and total red blood cell counts, total and differential white blood cell counts, haemoglobin, packed cell volume, mean corpuscular volume and mean corpuscular haemoglobin concentration were determined by routine Coulter S procedures. Serum and red blood cell concentrations of folate were determined by the method of Chanarin, Kyle & Stacey (1972), and serum cyanocobalamin concentration was determined according to Matthews (1962). Urine was collected for 24 h for the determination of glucaric acid excretion by the method of Marsh ( 1963), and for 9 h following the ingestion of a loading dose of histidine hydrochloride (15 g) for the determination of formiminoglutamic acid (FIGLU) by the method of Bennett & Chanarin (1961). The dietary intake for each patient was precisely recorded over 4 days during the collections of blood and urine samples. To ensure complete urine collections and accurate dietary records, patients were kept under close supervision for the duration of the study, but this did not involve any significant alteration of the patients' daily routine. The nutrient intake for each patient was calculated from standard food tables (McCance & Widdowson, 1960). Table I

Group

Sex distribution

Control Anticonvulsants Phenothiazines Tricyclics

8 + 7F 13M + 3F 10M + 5F 5M + 2F

Results The concentrations of folate of red blood cells and serum were significantly lower in patients receiving prolonged treatment with anticonvulsants (P < 0.0005 for cells and serum) and phenothiazines (P < 0.005 for cells, 10

169

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Figure 3 Correlation between urinary FIGLU and D-glucarate excretion. Controls are shown within the shaded area and patients treated with anti-convulsant drugs are ;r= 0.62. shown thus *

Duration of treatment (years) Figure 2 The urinary excretion of D-glucarate (mean mean) by control subjects and patients treated with enzyme-inducing drugs for long periods of time. + s.e.

long term anticonvulsant therapy urinary D-glucaric acid showed a significant negative correlation with urinary FIGLU excretion (r= 0.62) (Figure 3). No correlations were observed between patient age and the urinary excretion of D-glucaric acid, and there was no correlation between age and the duration of drug treatment.

Discussion

Although it is well established that megaloblastic haemopoiesis may occur as a complication of anticonvulsant therapy with phenytoin alone (Hawkins & Meynell, 1954), or primidone alone (Chanarin, Elmes & Mollin, 1958) or in combination with other drugs (Kidd & Mollin, 1957; Stokes & Fortune, 1958; Chanarin, Laidlaw, Loughridge & Mollin, 1960) none of the patients in the present study were found to be anaemic (Table 3). The absence of macrocytosis in our Table 4

patients is thus in marked contrast with the high incidence reported by Hawkins & Meynell (1958) (27-45% of 159 patients on phenytoin or phenobarbital alone, or in combination) and Klipstein (1964) (74% of 53 patients on phenytoin), but is in agreement with the findings of Reynolds et al. (1966) who did not detect macrocytosis in a study of 45 epileptics. The morphological abnormalities found in the blood cells of some of the patients we studied (Table 3) may reflect more specific side-effects of the drugs prescribed. However, the patients in the present study, receiving long-term treatment with anticonvulsant drugs or phenothiazines did show a high incidence of folate deficiency judged by the decreased serum and red blood cell folate concentrations (Table 2). The high incidence of decreased serum and red blood cell folate concentration in epileptic patients treated with drugs is well documented (Klipstein, 1964; Houben, Hommes & Knaven, 1971; Preece, Reynolds & Johnson,

Urinary excretion of D-glucarate and FIGLU following histidine loading in patients treated with anti-convulsant, phenothiazine and tricyclic drugs

Group

Number of

patients

Mean duration of treatment

FIGLU

D-glucarate

(mg/9 h)

(ilmo//24 hJ

(years) Controls Anticonvulsants Phenothiazines Tricyclics

15 16 15 7

12 15 2

7.1 21.7 16.5 10.5

+ 1.6 + 3.1

± 1.6 + 2.7

9.3 + 93.3 + 28.0 ± 32.5 +

1.7 17.6 5.4 7.2

DRUG ENZYME INDUCTION AND FOLATE DEFICIENCY

1971) but there is no similar evidence of folate deficiency resulting from long-term administration of phenothiazines. Despite the chemical, metabolic and pharmacological dissimilarities of anticonvulsants and phenothiazines our findings may be indicative of a common underlying mechanism in the precipitation of folate deficiency by long-term drug administration. Nevertheless, inadequacy of dietary intake of folate might contribute to the folate deficiency in the phenothiazine group as the ratios of serum and red cell folate to dietary intake in -this group did not differ significantly from controls. Folic acid deficiency in our patients was also assessed by the urinary excretion of FIGLU following histidine loading (Luhby, Cooperman & Teller, 1959). We found that 7 out of 14 patients receiving anticonvulsant drugs, 6 of 15 on phenothiazines, and 1 of 7 treated with tricyclic drugs, excreted amounts of FIGLU that were above the upper limit of the normal range (18 mg/24h). Moreover, the mean value of FIGLU excretion for each of the three groups of drugtreated patients was higher than the control group (Table 4) and this is indicative of sub-clinical folate insufficiency. However, in the case of the patients treated with tricyclic drugs the increase in FIGLU excretion did not reach levels of significance, possibly because of the shorter duration of treatment. Urinary FIGLU excretion above the upper limit of normal has been previously reported in 23% of epileptics treated with anticonvulsant drugs (Reynolds, et al, 1966). The increase in the urinary excretion of FIGLU in patients on long-term drug treatment was accompanied by a marked increase (4- to 10-fold) in the urinary excretion of D-glucaric acid (Table 4). The excretion of this acid, a product of the glucuronic acid pathway, has been used as an index of hepatic microsomal enzyme induction and is elevated following administration of enzyme inducing drugs (Breckenridge, 1975; Latham, Turner, Franklin & Maclay, 1976). Increased glucarate excretion has previously been associated with decreased serum and red blood cell folate concentrations in epileptics treated with anti-convulsants (Maxwell et al., 1972). In our patients, the increased excretion of glucarate was accompanied by an increase in FIGLU excretion and by a decrease in serum and red blood cell folate, both in epileptics treated with anticonvulsant drugs and in other patients treated with phenothiazines. In patients treated with tricyclic drugs, although the urinary excretion of glucarate was increased 3- to 4fold, the decreases in folate concentration and increase in excretion of FIGLU were insufficient to be significant. This is possibly due to the shorter period for which the drugs had been given, and it would seem that prolonged exposure to enzyme inducing agents is necessary before folic acid deficiency ensues (Table 4 and Figure 1). From a consideration of folate deficiency, measured by FIGLU excretion, as a function of the period of chronic administration, it became

171

evident that folic acid deficiency was present in patients who had received enzyme-inducing drugs for a period of 2-5 years, but was far more marked in those treated for longer periods of time (Figure 1). However, the enzyme inductive effects of the drugs, as measured by the urinary excretion of D-glucarate, was greatest (>10 fold) in those patients who had been treated for two-five years and, although still increased above normal, was less (4- to 5-fold) in those who had received drugs for a longer period (Figure 2). It seems therefore, that the degree of response to an enzymeinducing agent is related, at least in part, to the folate status. In folate deficiency, due to a dietary intake of folate inadequate to meet the increased requirements of enzyme induction, the de novo synthesis of microsomal enzymes may return to normal, or even diminish. Thus in patients on long-term treatment with anticonvulsants, the urinary excretion of D-glucarate shows a significant correlation with the folic acid status of these subjects (Figure 3). This may explain why patients, apparently stabilised for many years on a particular dose of anticonvulsant, suddenly develop symptoms of toxicity for no apparent reason (Figure 4). It has also been shown that dietary deficiency of folate in the rat results in impairment of enzyme induction as measured by the liver microsomal content of cytochrome P-450. Similarly, prolonged administration of phenobarbitone and phenytoin to rats on folate supplemented or folate semi-supplemented diets leads to folate deficiency, and dietary deficiency of folate with prolonged administration of drugs shows additive effects in producing folate deficiency and impairment of liver enzyme induction (Labadarios, 1975). If the folic acid deficiency observed in epileptic and other patients is indeed the result of increased utilization due to the induction of hepatic enzymes by drugs, then these patients would be expected to have an impaired capacity to metabolise drugs, and folate supplementation should be considered to restore or even augment their drug-metabolising capacity. During treatment with folic acid of patients suffering from phenytoin intoxication the metabolism of phenytoin was found to be accelerated (Kutt, Winters & McDowell, 1966). Similarly, the serum concentration of phenytoin decreases during folic acid supplementation of stabilised patients on long-term anticonvulsant therapy (Jensen & Olesen, 1970; Baylis, Crowley, Preece, Silvester & Marks, 1971). However, it has been suggested that the antifolate effect of anticonvulsant drugs may be related to their therapeutic action (Reynolds, 1967) for epileptic patients on prolonged anticonvulsant therapy, given high doses of folate (15-30 mg) for treatment of megaloblastosis, show a deterioration in seizure control (Chanarin et al., 1960; Reynolds, 1967). The requirement of man for folic acid is thought to be about 50-100 Rtg pteroylglutamic acid per day (DHSS, 1969), which probably refiects the minimum

172

D. LABADARIOS, J.W.T. DICKERSON, D.V. PARKE, E.G. LUCAS & G.H. OBUWA

Chronic administration of drugs

4

Enzyme induction 2-5 years

Increased requirement for folate

Increased drug metabolism

Reduced de novo synthesis of

drug-metabolizing enzymes > 5 years

Accumulated deficiency of folate

Normalisation or reduction of drug metabolism

Drug toxicity

Figure 4 Possible relationships of chronic drug administration, induction of drug-metabolising enzymes, and accumulating folate deficiency.

necessary for good health. The calculated daily intake of patients in the present study was about 50 pLg per day, except for the tricyclic group of patients, whose intake was significantly lower (Table 2) and well below the recommended intake. The dietary intake of folic acid by patients in other psychiatric hospitals appears to be about this latter value (Dickerson, unpublished observations). The folate intake of these patients is therefore, probably barely sufficient for normal metabolic requirements, and certainly inadequate to meet the increased folate utilisation caused by chronic dosage with drugs. The unequivocal establishment of a requirement of folate in man to facilitate the de novo protein synthesis required for liver enzyme induction might have been achieved by the administration of folate to those patients with more than five years drug treatment who exhibited impaired enzyme induction. However, this was considered to be unethical as the expected restoration of enzyme induction would have likely resulted in

reduced pharmacological activity of the prescribed dose of drug and disturbance of the therapeutic control of the patients. However, such evidence has been obtained in the rat (Labadarios, 1975). Nevertheless, in many hospitals folate supplementation is now being made but this has to be effected with care to avoid the sudden re-emergence of enzyme induction with consequent diminution of blood levels of the drugs. Folate supplementation is therefore carefully regulated with regard to regular monitoring of blood concentrations of both folate and drugs prescribed (Dickerson, personal communication). Moreover, recent studies have shown that folate deficiency does not occur in patients on long-term drug therapy where the dietary intake of folate is adequate (Dickerson, personal communication). Thanks are due to the Nursing and Catering staff of Brookwood iHospital for their cooperation during this study. DL was an MRC Research Scholar.

References BAYLIS, E.M., CROWLEY, J.M., PREECE, J.M., SYLVESTER, P.E. & MARKS, V. (1971). Influence of folic acid on blood phenytoin levels. Lancet, 1, 62-64. BENNETT, M.C. & CHANARIN, I. (1961). Urinary excretion of urocanic acid in megaloblastic anaemia. Lancet, ii 1095. BRECKENRIDGE, A. (1975). Clinical implications of enzyme induction. In Enzyme Induction, ed. Parkes, D.V. pp. 273-302. London: Plenum Press. CHANARIN, I., ELMES, P.C. & MOLLIN, D.L. (1958). Folic-acid studies in megaloblastic anaemia due to primidone. Br. med. J., 2, 80-82. CHANARIN, I., KYLE, R. & STACEY, J. (1972). Ex-

perience with microbiological assay for folate using a chloromphenicol resistant L. casei strain. J. clin. Path., 25, 1050-1052. CHANARIN, I., LAIDLAW, J., LOUGHRIDGE, L.W. & MOLLIN, D.L. (1960). Megaloblastic anaemia due to phenobarbitone. The convulsant action of therapeutic doses of folic acid. Br. med. J., 1, 1099-1102. DAHLKE, M.B. & MERTENS-ROSLER, E. (1967). Malabsorption of folic acid due to diphenylhydantoin. Blood, 30, 341-35 1. DEPARTMENT OF HEALTH AND SOCIAL SECURITY. (1969). Recommended intakes of nutrients for the United Kingdom. H.M.S.O.

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MANTZ, J. M., TEMPE, J.D. & JAEGAR, A. (1970). Intoxication barbiturique et anemie megaloblastique. Eur. J. Tox., 2, 130-137. MARSH, C.A. (1963). Metabolism of D-glucuronolactone in mammalian systems. Identification of D-glucaric acid as normal constituent of urine. Biochem. J., 86, 77-86. MATTHEWS, D.M. (1962). Observations on the estimation of serum vitamin B12 using Lactobacillus leichmannii, Clin. Sci., 22, 101-111. MAXWELL, J.D., HUNTER, J., STEWART, D.A. & WILLIAMS, R. (1972). Folate deficiency after anticonvulsant drugs: an effect of hepatic enzyme induction? Br. med. J., 1, 297-299. MEYNELL, M.J. (1966). Megaloblastic anaemia in anticonvulsant therapy. Lancet, i, 487. PREECE, J., REYNOLDS, E.H. & JOHNSON, A.L. (1971). Relation of serum to red cell folate concentrations in drug-treated epileptic patients. Epilepsia, 12, 335-340. REYNOLDS, E.H. (1967). Effects of folic acid on the mental state and fit frequency of drug-treated epileptic patients. Lancet, i, 1086-1088. REYNOLDS, E.H. (1968). Mental effects of anticonvulsants and folic acid metabolism. Brain, 91, 197-214. REYNOLDS, E.H., MILNER, G., MATTHEWS, D.M. & CHANARIN, I. (1966). Anticonvulsant therapy, megaloblastic haemopoiesis and folic acid metabolism. Quart. J. Med., 35, 521-537. RICHENS, A. (1976). Liver enzyme induction by antiepileptic drugs: its clinical significance. In Anticonvulsants Drugs and Enzyme Induction, eds. Richens, A. & Woodford, F. P., pp. 3-12. Amsterdam: Elsevier. ROSENBERG, I.H., STREIGG, R.R. & GODWIN, H.A. (1968). Impairment of intestinal deconjugation of dietary

folate. (A possible explanation of megaloblastic anaemia associated with phenytoin therapy). Lancet, iL 530-532. STOKES, J.B. & FORTUNE, C. (1958). Megaloblastic anaemia associated with anticonvulsant drug therapy. Australas. Ann. Med., 7, 118-125. TIETZ, A., LINDBERG, M. & KENNEDY, E.P. (1964). A new pteridine-requiring enzyme system for the oxidation of glyceryl ethers. J. biol. Chem., 239,4081-4090.

(Received February 25, 1977)

The effects of chronic drug administration on hepatic enzyme induction and folate metabolism.

Br. J. clin. Pharmac. (1978), 5, 167-173 THE EFFECTS OF CHRONIC DRUG ADMINISTRATION ON HEPATIC ENZYME INDUCTION AND FOLATE METABOLISM D. LABADARIOS*,...
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